The overall goal of this study is to address the therapeutic potential of cyc E siRNA on the treatment of hepatocellular carcinoma (HCC). Our lab recently discovered that cyclin E (cyc E), an oncogene overexpressed in 70% of HCC, played a substantial role in proliferation and cell survival and could serve as a promising therapeutic target for HCC. We also found that overexpressed cyc E could be suppressed up to 90% by siRNA targeting the coding region of cyc E. Depletion of Cyc E in HCC induced significant inhibition of cell growth both in cultured cells and in nude mice. Therefore, we hypothesize that cyc E siRNA may serve as a novel and effective therapeutic agent to treat cyc E-overexpressing HCC. Four specific aims will be carried out to test this hypothesis. (1) To determine the therapeutic effects of cyc E siRNA using both subcutaneous and orthotopic HCC models in mice. For the orthotopic model, HCC cell lines expressing luciferase will be intrahepatically injected into mice to produce tumors in liver. An improved liposomal delivery system (DOTAP:Chol) will be used for siRNA transfer by intratumoral injection or systemic treatment through intravenous injection. The tumor volume and metastasis will be monitored by in vivo image system to determine the therapeutic efficacy. We will also compare the tumor suppression effects among treatments with different delivery systems (non-liver targeting versus liver-targeting delivery system) to develop an optimal preclinical therapeutic strategy for HCC. (2) To examine the effect of cyc E siRNA on HCC cells versus normal hepatocytes or tissues in in vitro and in vivo models. The siRNA will be transfected into immortalized normal human hepatocytes or HCC cells and the growth properties among those cells will be fully analyzed for their differences. The in vivo toxicity of siRNA will be examined by enzymatic and pathological analysis on major organs in mice after the treatments. (3) To evaluate the therapeutic efficacy of cyc E siRNA in combination with chemodrugs. We have demonstrated that combination of cyc E siRNA and doxorubicin exhibited a synergism on inhibition of HCC cell growth. To test if cyc E overexpression is involved in chemoresistance, stable cell lines with different cyc E expression levels will be generated and examined for their responses to multiple chemodrugs. The synergistic effects from the combinations involving cyc E siRNA and chemodrugs will be further tested in animals. We will also identify the mechanisms mediating these synergistic effects, including analysis of NF-kappaB, Akt and Bcl2 survival pathways. (4) To assess the in vitro and in vivo antitumor effect of cyc E siRNA from a tumor specific expression vector. We have generated a plasmid which expresses cyc E siRNA via a liver tumor specific promoter (AFP). The efficacy and specificity of this vector will be systematically assessed using both HCC cell lines and HCC xenograft models in mice. The data generated from this study will provide valuable information to further understanding the molecular events involved in the development of HCC as well as lead to the development of effective cyc E siRNA-based therapy to reduce HCC mortality.